Using Lentiviral shRNA Delivery to Knock Down Proteins in Cultured Neurons and In Vivo

Wilkinson, Kevin A.; McMillan, Kirsty J; Banks, Paul J; Carmichael, Ruth E.; Nakamura, Yasuko; Bashir, Zafar; Cullen, Peter J.; Henley, Jeremy M

doi:10.1007/978-1-0716-2569-9_1

Cited by 5 publications

(8 citation statements)

References 30 publications

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“…To further investigate GluK2 palmitoylation and its interplay with other post-translational modifications, we used a lentiviral GluK2 shRNA knockdown (KD) and replacement strategy in rat cortical neuronal cultures ( Wilkinson et al, 2022 ). We engineered lentiviral constructs that knocked down endogenous GluK2 and simultaneously expressed shRNA-insensitive YFP-myc-tagged GluK2 variants.…”

Section: Resultsmentioning

confidence: 99%

“…To conduct GluK2 knockdown experiments, shRNA sequences specifically targeting GluK2 were inserted into a modified pXLG3-GFP vector ( Wilkinson et al, 2022 ), under the control of an H1 promoter. The shRNA target sequences used were as follows:…”

Section: Methodsmentioning

confidence: 99%

“…Control, non-targeting shRNA (SCR): AATTCTCCGAACGT GTCAC GluK2-targeting shRNA (SH1): GCCGTTTATGACACTTGGA For knockdown-replacement experiments, N-terminally YFP-myc-tagged rat GluK2 (WT or indicated mutants) containing five silent point mutations rendering them insensitive to the GluK2 shRNA were cloned into the pXLG3-GluK2 shRNA plasmid, in place of the GFP. The viral particles containing these constructs were generated in HEK293T cells as described previously (Wilkinson et al, 2022). Subsequently, the harvested viral particles were added to cortical neurons at 12-14 days in vitro (DIV) and allowed to incubate for 7 days before being used for further experiments.…”

Section: Transduction With Lentivirusmentioning

confidence: 99%

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Coordinated interplay between palmitoylation, phosphorylation and SUMOylation regulates kainate receptor surface expression

Yucel,

Al Momany,

Evans

et al. 2023

Front. Mol. Neurosci.

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Kainate receptors (KARs) are key regulators of neuronal excitability and synaptic transmission. KAR surface expression is tightly controlled in part by post-translational modifications (PTMs) of the GluK2 subunit. We have shown previously that agonist activation of GluK2-containing KARs leads to phosphorylation of GluK2 at S868, which promotes subsequent SUMOylation at K886 and receptor endocytosis. Furthermore, GluK2 has been shown to be palmitoylated. However, how the interplay between palmitoylation, phosphorylation and SUMOylation orchestrate KAR trafficking remains unclear. Here, we used a library of site-specific GluK2 mutants to investigate the interrelationship between GluK2 PTMs, and their impact on KAR surface expression. We show that GluK2 is basally palmitoylated and that this is decreased by kainate (KA) stimulation. Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation and K886 SUMOylation under basal conditions and is insensitive to KA-induced internalisation. These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Transduction With Lentivirusmentioning

confidence: 99%

See 1 more Smart Citation

Coordinated interplay between palmitoylation, phosphorylation and SUMOylation regulates kainate receptor surface expression

Yucel,

Al Momany,

Evans

et al. 2023

Front. Mol. Neurosci.

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“…Specifically, human Tau 4R0N isoform WT or P301L coding sequences were amplified from pSinRep5-EGFP-Tau-expressing plasmids (a gift from Prof. Neil Marrion, University of Bristol, UK) by PCR and cloned with an N-terminal Myc-tag into the SpeI and BamHI sites of the lentiviral plasmid pXLG3-PX-WPRE ( Wilkinson et al, 2022 ).…”

Section: Methodsmentioning

confidence: 99%

Aggregation-prone Tau impairs mitochondrial import, which affects organelle morphology and neuronal complexity

Needs

Wilkinson

Henley

et al. 2023

Journal of Cell Science

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Mitochondrial protein import is essential for organellar biogenesis, and thereby for the sufficient supply of cytosolic ATP–particularly important for cells with high energy demands like neurons. This study explores the prospect of import machinery perturbation as a cause of neurodegeneration instigated by the accumulation of aggregating proteins linked to disease. The aggregation-prone Tau variant (TauP301L) reduces the levels of components of the import machinery of the outer (TOM20) and inner membrane (TIM23) while associating with TOM40. Intriguingly, this interaction affects mitochondrial morphology, but not protein import or respiratory function; raising the prospect of an intrinsic rescue mechanism. Indeed, TauP301L induced the formation of tunnelling nanotubes (TNTs), potentially for the recruitment of healthy mitochondria from neighbouring cells and/or the disposal of mitochondria incapacitated by aggregated Tau. Consistent with this, inhibition of TNT formation (and rescue) reveals Tau-induced import impairment. In primary neuronal cultures, TauP301L induced morphological changes characteristic of neurodegeneration. Interestingly, these effects were mirrored in cells where the import sites were blocked artificially. Our results reveal a link between aggregation-prone Tau and defective mitochondrial import machinery relevant to disease.

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“…Lentivirus was produced in HEK293T cells following standard protocols (42). HEK293T cells plated in 6-or 10-cm dishes were transfected using plain DMEM containing 2 μg/mL of the appropriate pXlg3 viral vector, 0.5 μg/mL pMD2.G, 1.5 μg/mL p8.91, and 12 μg/mL PEI in plain DMEM media for 4 hours.…”

Section: Lentivirus Production and Transduction Of Cortical Neuronsmentioning

confidence: 99%

SGIP1 binding to the α-helicalH9domain of cannabinoid receptor 1 promotes axonal surface expression

Fletcher-Jones,

Spackman,

Craig

et al. 2023

Preprint

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Endocannabinoid signalling mediated by cannabinoid type 1 receptors (CB1Rs) is critical for homeostatic neuromodulation of both excitatory and inhibitory synapses. This requires highly polarised axonal surface expression of CB1R, but how this is achieved remains unclear. We previously reported that the H9 domain in the intracellular C-terminus of CB1R contributes to polarised surface expression by an unknown mechanism. Here we show the H9 domain binds to the endocytic adaptor protein SGIP1 to promote CB1R expression in the axonal membrane. Overexpression of SGIP1 increases CB1R axonal surface localisation but has no effect on CB1R lacking the H9 domain (CB1RΔH9). Conversely, SGIP1 knockdown reduces axonal surface expression of CB1R but does not affect CB1RΔH9. Furthermore, SGIP1 knockdown diminishes CB1R-mediated inhibition of presynaptic Ca2+influx in response to neuronal activity. Together, these data advance mechanistic understanding of endocannabinoid signalling by demonstrating that SGIP1 interaction with H9 underpins axonal CB1R surface expression to regulate presynaptic responsiveness.

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Using Lentiviral shRNA Delivery to Knock Down Proteins in Cultured Neurons and In Vivo

Cited by 5 publications

References 30 publications

Coordinated interplay between palmitoylation, phosphorylation and SUMOylation regulates kainate receptor surface expression

Coordinated interplay between palmitoylation, phosphorylation and SUMOylation regulates kainate receptor surface expression

Aggregation-prone Tau impairs mitochondrial import, which affects organelle morphology and neuronal complexity

SGIP1 binding to the α-helicalH9domain of cannabinoid receptor 1 promotes axonal surface expression

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